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Appendix E: Panelists’ Comments Submitted After the Meeting

Historical Document

This Web site is provided by the Agency for Toxic Substances and Disease Registry (ATSDR)
ONLY as an historical reference for the public health community. It is no longer being maintained and the data
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The expert panelists were asked to provide premeeting comments
and to participate in the discussions at the expert panel review meeting.
In addition, several panelists chose to submit additional written comments
after the expert panel review meeting. Some panelists submitted updated
versions of their premeeting comments (see Appendix
B), while others wrote summaries of the discussions they led at the
expert panel review meeting. All post-meeting comments are presented here,
regardless of their content. Panelists were not required to submit post-meeting
comments.

This section presents the post-meeting comments exactly as they were
submitted to ERG, with only minor changes to format and references. The
expert panel was not asked to comment on the content of these post-meeting
comments.

Note:

Dr. Case submitted post-meeting comments as a list of suggested
revisions to an earlier draft of this report. Dr. Case’s comments have
been incorporated directly into the text of this report and are not replicated
here.

Dr. Lippmann’s Post-Meeting Comments

Topic # 1. Physiological Fate of Asbestos and Vitreous Fibers less
than 5 Microns in Length. Discuss/review current knowledge about the
physiological fate of small fibers when they enter the body.

A. What is the expected physiological depositional pattern for less-than-5-micron
fibers in the lung?

This is well established in terms of the depositional mechanisms of impaction,
sedimentation, Brownian motion and (for fibers) interception. Fibers with aspect
ratios >10 behave aerodynamically like unit density spheres with diameters
three times their fiber width (Stöber et al., 1970; Timbrell, 1972). The
only exception, in terms of being influential in deposition in lung airways
is for fibers longer than about 10 µm, where the mechanism of interception
becomes influential (Sussman et al, 1991). This also accounts for the fact that
longer fibers have proportionately more deposition in the airways as opposed
to peripheral alveoli. The fact that lung retention also increases more markedly
with fibers greater than 10 microns is supported by theoretical calculations
(Yu et al., 1990), analysis of lung dust content in humans (Timbrell, 1982;
Churg and Wiggs, 1987; Pooley and Wagner, 1998) and studies using experimental
animals (Morgan 1979, 1995). Thus, for fibers <5 µm in length, deposition
patterns and efficiencies will be determined almost entirely according to the
fiber width, which for fibers <5 µm long will be less than about 1.6
µm. For fiber widths between about 0.1 and 1.6 mm, total lung deposition
in healthy people will be between 10 and 20%, with almost all of it in the deep
lung. For fibers thinner than 0.1 µm, deposition will increase with decreasing
width, and there will be a somewhat greater proportion of the deposition in
the more proximal airways. Particles that are not deposited remain suspended
in the tidal air and are exhaled.

There are significant differences between humans and rats with respect to deposition
efficiencies of long as well as short fibers; respirability is very different
and the deposition fractions are significantly different as well between the
two species.

B. What is known about clearance/biopersistence of less-than-5-micron fibers
once deposited in the lungs?

For these short fibers, which can be fully engulfed by lung cells and do not
dissolve in airway fluids in less than a few weeks, their clearance will be
similar to other mineral and vitreous particles. Those depositing in lung conductive
airways will be largely removed to the G.I. tract by mucociliary clearance within
about one day. Most of those depositing in the gas-exchange region will be phagocytized
by alveolar macrophages and cleared to and through the mucociliary escalator
within a few weeks. Other particles may be engulfed by epithelial cells, primarily
in the vicinity of the bronchial-alveolar duct junctions, and retained for much
longer periods, with gradual removal to lymph nodes.

The relatively rapid clearance of short fibers and compact particles from the
lung has been demonstrated in a number of studies (reviewed in Health Effects
Institute-Asbestos Research, 1991; Davis, 1994; Oberdörster et al., 1990;
Morgan, 1995). Such particles can be: 1) readily transported through tracheobronchial
and other lymph nodes to more distal lymphatics, the pleura, or other organs;
2) cleared via the mucociliary escalator and alveolar macrophages; and 3) effectively
phagocytized by a number of cell types in the lung including epithelial cells
(Churg et al., 2000). Once within a phagolysosome or in general in lung fluids,
shorter fibers of chrysotile asbestos (Hume and Rimstidt, 1992) or glass (reviewed
in Lippmann, 1990) are more prone to dissolution and fragmentation than longer
fibers and amphibole types of asbestos.

Absent abnormalities in phagocyte function of these particles should be removed
even if they are chemically resistant if: (a) the dose is not too great to overwhelm
these normal mechanisms; and (b) the mechanisms themselves are intact. There
are medical conditions which affect these mechanisms, however, so there are
likely to be vulnerable populations (such as those with primary ciliary
disorders; these tend to be genetic and very rare such as primary ciliary
dyskinesia (incidence 1:20,000 to 1:60,000)). Of greater frequency is the lesser
effect on mucociliary clearance in asthma. In addition environmental influences,
including smoking and nitrogen dioxide (Case et al., 1982), can affect these
normal mechanisms through direct ciliary damage or disrupted function. Some
common pharmaceuticals slow mucociliary transport (for example, some general
anaesthetics and atropine), while others accelerate it (for example, theophyllines
and sympathomimetics). Bronchial secretion is also an important contributor
to clearance or impaired clearance, as can be seen most dramatically in cystic
fibrosis. Overall, then, there are a number of possible factors that may interfere
with particle clearance, but none have been associated with “fiber length” parameters
with the possible exception of smoking (Takahashi et al., 1994).

The most important physiological clearance mechanism in alveolar region is
clearance by alveolar macrophages (AM). Of importance is fiber length with respect
to phagocytosis and removal by alveolar macrophages. Short fibers are easily
phagocytized, fibers longer than 20 µm are not. There are species differences
in AM size. Thus, clearance for long fibers is prolonged, as is that for short
fibers when high lung burdens are reached (particle overload). Also, intrinsic
toxicity, which influences clearance, has to be considered. Inflammatory conditions
in the lung (for example, smokers) also contribute to impairment of alveolar
macrophage-mediated mechanical clearance and need to be considered.

Biopersistence is the sum of physiological clearance processes and physicochemical
processes, which together account for the retention halftime of the fibrous
or non-fibrous material in the lung. Physicochemical processes include dissolution,
leaching, breaking and splitting, depending on the fibrous material, that can
occur intra- as well as extra-cellularly, and differences in pH in both locations
are of importance here. Clearance rates of fibers of different length categories
have been determined from short- and long-term inhalation studies (Davis et
al., 1986, 1987; Wagner, 1990). Generally, short fibers are cleared rapidly
if biosoluble (pH differs intracellularly vs. extracellularly), or at rates
similar to nonfibrous particles. Breakage of long fibers will give input into
short fiber category.

The hazards associated with man-made vitreous fiber (MMVF) appear to be most
strongly associated with the ability to persist within lung tissue. This is,
in part, dependent upon chemical composition of the MMVF, in that increased
concentrations of stabilizers such as aluminum impact a greater degree of chemical
durability. In vitro tests to measure fiber solubility should be performed
to reflect an acid pH of 4.5 to 5.0 such as found in phagolysomes within alveolar
macrophages as well as pH of 7.4 reflecting extra-cellular fluid. Short fibers
that are ingested by macrophages will encounter the lower pH that overall could
affect their biopersistence. In general, solubility tests identified the following
rank order from lowest to greatest solubility of MMVF in comparison to asbestos
fibers: crocidolite <amosite <RCF <special purpose glass fibers <rock
wool <slag wool <conventional glass fibers (NRC, 2000).

In rodent exposure to mixed dust resulted in an increased transport of fibers
across the visceral pleura and increase production of lung tumors and mesothelioma
(IARC# 140, 1996).

C. What type(s) of migration are expected within the body for less-than-5-micron
fibers?

Fibers with diameters less than ~0.1 µm, which could be a significant
fraction of fibers <5 µm in length, can penetrate through the respiratory
epithelia and be transported through lymph channels to hilar and peripheral
(mesothelial) lymph nodes and through blood to more distant body organs. Gelzleichter
et al. (1996) exposed rats to nose only inhalation of kaolin-based refractory
ceramic fiber. It was identified that fibers rapidly translocate to the pleural
tissue with a difference between those in the pleural tissue and the parenchymal
tissue. Within the pleural tissue the geometric mean length 1.5 µm (GSD
~ 2.0) and geometric mean diameter 0.09 µm (GSD ~1.5). For comparison
parenchymal tissue GML = 5.0 µm (GSD ~2.3) and GMD 0.3 µm (GSD ~1.9.)
This would indicate the short thin fibers are capable of translocating to the
pleural tissue.

This may be an important subject, at least for the parietal pleura, if it is necessary for fibers to reach the pleura to cause lesions (plaques and
mesothelioma). It remains possible that fibers still within the peripheral lung
may be capable of contributing to the mechanisms of these diseases. Mechanisms
remain speculative, but long amphibole fibers may tend to localize toward the
lung periphery, and it remains possible (but unproven and indeed untested) that
chemical mediators may cross the visceral pleura into the pleural space. Churg
and Wiggs (1987), among others, have observed that “accumulation of long fibers
immediately under the upper lobe pleura may be important in the genesis of mesothelioma.”

Two recent studies are informative (Boutin et al., 1996; Dumortier et al.,
2002). They found that “the distribution of asbestos fibers in the pleura was
heterogeneous and that they might concentrate in…’black spots’ of the parietal
pleura.” Using thoracoscopy in living patients from “normal areas of the parietal
pleura” rather than plaques and tumor, and using controls, they showed that
“amphiboles outnumbered chrysotile in all samples” and that of all fibers 22.5%
were in fact greater than or equal to 5 µm in length; a proportion at
least as great as that usually seen in lung tissue. The means of translocation
remains unknown, although these findings strongly suggest lymphatic drainage
paths. The pathogenic significance also remains unknown, although the authors
emphasized their hypothesis that these fibers might contribute to plaque and
mesothelioma genesis.

Other papers that have been published (in relation to human disease) have been
for the most part based on static “fiber burdens” that purport to be in “the
pleura” but which on careful reading are in fact in mesotheliomatous tissues
and/or pleural plaques; the false assumptions are then made that “short fibers”
- usually very short chrysotile fibers, averaging less than 0.2 µm in
length - have “translocated” to the “pleura” from the lung. In fact the
“pleura” was not studied, tumor and plaque, which by definition could
not contain fibers except via specimen contamination or incorporation, most
likely from adjacent lung. Both Rogers et al. (1994) and Case et al. (1994)
have also reported contamination by short crocidolite fibers of Nuclepore
filter materials and in uncontrolled studies of this nature any material from
air, fluids, and paraffin in the pathology laboratory from which the specimens
originally were referred to specimen preparation materials are suspect.

Dr. Lockey’s Post-Meeting Comments

What do human/epidemiological data tell us about small fibers? Discussion Leaders: Dr. Lockey and Dr. Case

Cancer Effects

Short natural occurring fibers. A study by Higgins, et al. [1] in 1983
reviewed the mortality of workers employed at the Reserve Mining Company at
Babbit, Minnesota. These workers were involved with mining taconite, which is
a dense hard rock composed of silica, silicates and iron. Taconite mined in
the eastern tip of the Mesabi range contained amphiboles in the cummingtonite-grunerite
series. These fibers are short in length with reportedly the vast majority being
<10 µm and are related to amosite asbestos. Of the 9,065 men employed
by the company as of July 1, 1976, 5,751 had worked one year or more. The investigators
established the vital status of 96% of those who worked for five years or longer
and 75% of former workers who worked one to four years. The total respirable
dust ranged from 0.02 mg/m3 to 2.52 mg/m3 and as high
as 2.75 mg/m3 with the modal range from 0.2 mg/m3 to 0.6
mg/m3. There were relatively few measurements of fibers and those
that were available demonstrated concentrations usually low with a few at or
above 0.5 fibers/ml in the crushing department. Reportedly none approached the
OSHA threshold limit value which at that time was 2 fibers/ml. Results of the
study indicated that there was no excess death in this population including
those men with cumulative exposure of 1,000 to 3,000 total dust years or 500
to 1,000 silica dust years. The conclusions of the study indicated the death
rates for all causes were significantly below expectations including selected
respiratory disease and death from malignant disease was marginally below that
expected for the State of Minnesota. There was no relationship between lifetime
dust exposure and increased mortality, nor was there any indication that malignant
neoplasm was increased after 15 to 20 years latency. The authors identified
a weakness of the study in that the average latency of the cohort was 14.7 years
with a maximum of 24.6 years, or a relatively short latency for development
of cancer.

A study by McDonald, et al. [2] regarding the mortality from long-term exposure
to cummingtonite-grunerite from a gold extraction process at the Homestake Mine,
Lead, South Dakota was reviewed. Those workers who had worked 21 years or longer
were traced and of 660 men who had died, the cause of death was ascertained
for 657. Results of the study indicated pneumoconiosis, which was mainly silicosis
along with tuberculosis, and heart disease were causes of excess death. There
was a dust exposure relationship for both pneumoconiosis and respiratory tuberculosis,
but reportedly no convincing increase in respiratory cancer. It was noted that
more than 75% of the 660 men who had died started to work before 1925. The interval
between first employment and death and the 76 fatalities from tuberculosis or
pneumoconiosis ranged from 22 to 61 years with a median of 35 years. Average
silica dust concentrations ranged from 11.0 to 24.6 mppcf before 1952. A study
by Dement, et al. [3] reported that 80% to 90% of fibers in the mine had an
amphibole diffraction pattern by transmission/scanning electron microscope equipped
with an energy-dispersive X-ray spectrometer. The mean total fiber concentration
was 4.82 ± 0.68 f/cc (range 0.66–11.79) with 0.36 ± 0.08 f/cc
(range 0.07-1.29) greater than 5 µm in length. There was one potential
mediastinal mesothelioma which could not be confirmed in the 17 respiratory
malignancies (16.5 expected based on South Dakota rates). The results of the
study were in conflict with an earlier study by Gillam, et al of the same mine
of 440 males who worked at least five years underground by 1960. Reportedly
there were 10 deaths from neoplasm of the respiratory system between 1960 and
1973 where as 2.7 were expected based on the male population of South Dakota
[4].

Conclusions

There is no data regarding human exposure to asbestos fiber uniformity less
than 5 µm in length.

Studies of workers exposed to cummingtonite-grunerite, a type of amphibole
related to amosite, demonstrated no consistent increase in overall mortality,
mortality related to selected respiratory disease, or respiratory cancer.
The vast majority of airborne fibers were reported to be less than 10 µm
in length.

Studies of workers of a gold mine in Lead, South Dakota exposed to cummingtonite-grunerite
initially demonstrated an increased mortality from malignant respiratory disease.
A subsequent study did not confirm the initial finding but demonstrated an
increase in silicosis and tuberculosis. Mean total fiber concentration was
4.82 f/cc with 0.36 f/cc greater than 5 µm in length.

Consideration should be given for performing a feasibility study regarding
an updated mortality analysis of these two cohorts.

MMVF Mortality Studies. Mortality studies of glass fiber and mineral wool production
workers have been ongoing in the U.S. most recently under the direction of Marsh,
et al at the University of Pittsburgh, and within the European Union under the
direction of the International Agency for Research on Cancer (IARC). The most
recent follow up study by Marsh, et al. [5,6,7] of 10 U.S. glass fiber manufacturing
plants demonstrate no excess mortality from all causes, all cancers combined,
or non-malignant respiratory disease. For respiratory system cancer, there was
an observed 6% excess that was statistically significant for the total cohort
but not found in workers who had five or more years of employment. An association
was seen with calendar time and time since first employment, but no relationship
was found with duration of employment, or increase in exposure to respirable
glass fiber. A case-control study of respiratory system cancer did not identify
increased risk with duration of exposure, cumulative exposure, or time since
first employment. An association with non-baseline levels of average intensity
of exposure to respiratory fibers was not present when adjusted for smoking.

A previous case-control study of a glass fiber manufacturing facility included
in the U.S. glass fiber study demonstrated that differences in local versus
national smoking rates may have been a contributing factor in the excess respiratory
cancer seen in that manufacturing facility. [8] The potential confounding impact
of cigarette smoking in the U.S. glass fiber and rock/slag wool studies was
further explored by Buchanich, et al. [9] and Marsh, et al. [10] and identified
as the potential unaccounted for factor regarding the small excess respiratory
system cancer not related to exposure indices.

Previous analysis of five rock and slag wool plants in the U.S. demonstrated
increased lung cancer mortality using U.S. but not local rates, and this was
confined to short-term workers or those workers with less than five years duration
of employment. There was no association with measures of respirable fiber exposure.
[11] Within the U.S. a case-control study of 9 slag wool plants demonstrated
an association with smoking but not MMVF exposure. [12]

Most recent analysis of the U.S. rock and slag wool workers as well as glass
fiber production workers identified ten death certificates that mentioned the
term mesothelioma. [13] Of the ten cases of mesothelioma, two on pathology review
were definitely not felt to be mesotheliomas, one had a 50% chance of mesothelioma,
and two others had less than 50% chance of mesothelioma. Medical records or
pathology specimens were not available on the remaining five. Using a timeframe
when specific malignant mesothelioma coding rubrics were available, the expected
mesothelioma rate (local county comparison) was 2.19 versus 1 observed. Overall
the authors felt there was no increased risk from the malignant mesothelioma
in the U.S. MMVF cohort.

The IARC have followed the mortality of workers among 13 MMVF manufacturing
facilities in Europe. [14] The most recent update demonstrated a significant
increase in lung cancer mortality in rock and slag wool workers as well as glass
wool workers, using national mortality rates which disappeared for the glass
wool workers when using local adjustment factors to the national mortality rates.
In addition, there was no association in the glass wool workers with time since
initial employment or duration of employment, and with removal of glass wool
workers with less than one-year employment no excess lung cancer was noted.

Within the rock and slag wool cohort there was an increase in lung cancer risk
but the authors felt there was no clear information to indicate that the increased
cancer risk was specifically related to fiber exposure. [14] A subsequent cohort
study demonstrated similar results. [15] A case-control study nested in this
latter cohort indicated no relationship between cumulative rock or slag wool
exposure and lung cancer. [16,17]

Within the IARC study there were five cases of mesothelioma, two which occurred
in workers with less than one-year employment and two in workers with most likely
prior asbestos exposure. [14]

Preliminary results of a mortality study of U.S. RCF manufacturing workers
demonstrate no significant increase in malignant or non-malignant respiratory
mortality and no malignant mesothelioma. The power of the study was limited
as the cohort was relatively young and small in number. [18]

Conclusions

There are no data regarding human exposure to MMVF uniformity less than
5 µm in length.

There is no persuasive evidence that exposure to glass fiber, rock wool,
slag wool, or refractory ceramic fiber has been associated with increased
lung cancer risks based on ongoing U.S. and European mortality studies.

There is no indication of an increased risk for mesothelioma.

Non-Cancer Effects

MMVF Morbidity and Mortality Studies. Non-malignant respiratory effects: Studies
of five fiberglass and two mineral wool manufacturing facilities identified
small opacities in 1.6 % of the population studied that were predominantly irregular
in shape. [19] These workers were involved with working in facility manufacturing
fibers over 3 µm in diameter and fibers averaging 1 µm to 3 µm
in diameter. The overall rate of chest X-ray changes was no different in comparison
to a non-MMVF exposed comparison group, and any relationship between exposure
indices was seen at profusion level 1/0 but not 1/1. There was no increase in
upper or lower respiratory tract symptoms. Similar results were seen in a study
in Australia of glass and rock wool production workers with no findings of asthma,
pulmonary fibrosis or pleural disease. [20] A similar study of rock wool workers
also did not demonstrate increased respiratory symptoms or abnormalities with
DLCO or DL/Va. A potential additive or synergistic effect, however, was seen
regarding the FEV1/FVC ratio, fiber exposure, and those with greater than 40-pack
year history of cigarette smoking. [21]

The IARC [22] study demonstrated no increased mortality from asthma, bronchitis
or emphysema, which is similar to the most recent analysis of the glass fiber
workers in the United States which did not identify increased mortality from
non-malignant respiratory disease. [5] Of interest in the IARC study was the
suggestion of an increased risk from non-malignant renal disease (SMR 0.97,
95% CI 0.36 to 2.11) in regard to duration of employment or employment at an
early phase within the rock and slag wool industry. Within the U.S. mineral
wool study a similar trend was noted (p < .05) with a SMR of 204 (observed
12) in regard to nephritis and nephrosis. [11] Similar type patterns have not
been demonstrated in relationship to nephritis and nephrosis deaths in U.S.
glass wool manufacturing facilities. [23]

There are very limited studies on end users of man-made vitreous fibers. One
study identified increased prevalence of chest radiograph evidence of irregular
opacities in workers using rotary spun fiberglass, but there was a question
of airborne asbestos fibers within the plant site. [24,25] In insulators a decrease
in FEV1 was identified in comparison to a non-exposed control group after adjusting
for smoking habits and self-assessed former asbestos exposure. [26]

On-going morbidity studies of workers involved with refractory ceramic fiber
(RCF) manufacturing have identified a relationship between pleural plaques and
time from initial employment, duration of employment, and cumulative refractory
ceramic fiber exposure. Pleural changes were seen 2.7% or 27 workers out of
1,008 of which 22 were pleural plaques. Of those with greater than 20 years
latency from initial production job or 20 years duration in a production job,
16 workers or 8.0% and 5 workers or 8.1% had pleural changes, respectively.
Interstitial changes were noted in 1.0% at profusion category >1/0, similar
to other non-specified dust exposed worker populations and showed a non-significant
elevated OR in regard to cumulative fiber exposure of 4.7 (95% CI, 0.97 to 23.5).
In regard to cumulative fiber exposure, 5.4% (8 of 148) with greater than 45
to 135 fiber-month/cc exposure had pleural changes (OR 5.6, 95% CI, 1.5 – 28.1).
For those with >135 fiber-months/cc exposure, 9.8% (6 of 61) had pleural
changes (OR 6.0, CI 1.4 – 31.0). [27] European studies concurred that there
was some evidence of a relationship between RCF latency and pleural changes
including pleural plaques but not duration or intensity of RCF exposure, but
it was difficult to separate the effects asbestos and RCF exposure and any relationship
between RCF exposure and small opacities was at best ambiguous. [28]

Previous studies of the RCF workers demonstrated a relationship between 10
years of employment in production job tasks prior to 1987 and small decrements
in FVC for current (165.4 ml) and past (155.5 ml) male smokers, but not never-smokers,
and small decrements in FEV1 for current male smokers only (134.9 ml). For never-smoker
women there was also a decrement in FVC (350.3 ml) per 10-years employment in
production job tasks. [29] A longitudinal analysis in those male workers able
to provide five tests or more did not demonstrate any further decrement of the
FEV1 or FVC between initial and final tests. [30]

Conclusions Regading Non-Cancer Effects of MMVF

There are no available morbidity studies of workers exposed to MMVF uniformly
less than 5 µm in length.

No increased mortality from non-malignant respiratory disease.

No indication of chest radiograph interstitial or pleural changes in regard
to glass and mineral wool production workers but data is limited.

Refractory ceramic fiber (RCF) exposure appears to be associated with the
occurrence of pleural plaques that most likely are related to increased exposure
levels in the RCF manufacturing facilities prior to 1985.

Potential additive or synergistic effect with MMVF exposure and small decrement
in FVC and/or FEV1 involving current or former smokers.

Within mineral wool cohort, question of potential increased mortality from
non-malignant renal disease such as nephritis and nephrosis.

End user studies of MMVF users are limited and are confounded by potential
previous asbestos exposure.

Irritant Effects

MMVF can cause skin irritation particularly in an area where clothing comes
in close contact to the skin such as around the neck or forearms. Essentially
this occurs in 5% of new workers involved with MMVF production. [31] Residential
contamination of man-made vitreous fibers in high concentration can also cause
irritation to the upper as well as lower respiratory tract. [32] Glass fibers
with diameters greater than 5.3 µm have been reported to be more likely
to cause skin irritation than the smaller diameter fibers, mainly due to mechanical
irritation. [33,34] There has been documentation of eye irritation associated
with MMVF as well as nasal and pharyngeal irritation with unusual MMVF dust
exposure situations. [35,36]

Conclusions

Skin irritation appears to be related to the mechanical effects of fiber
~5 µm in diameter and appears to be worse in hot, humid weather.

Accidental exposure to increased concentrations of MMVF can result in upper
and lower respiratory tract irritation as well as eye irritation.

Association Between Fiber Length and Fiber-like Toxicity

There have been no published studies that address whether asbestos fibers uniformly
<5 µm in length have been associated with pleural or parenchymal disease
in human. Any potential risk associated with fiber exposure <5 µm in
length most likely would be related to an increased risk for pulmonary asbestosis,
and most likely would occur at a substantially higher dose in comparison to
exposures to asbestiform fibers (long fibers with high aspect ratios). [37]

There is some indication that fibers with diameters with <0.1 µm to
0.4 µm and lengths <10 µm may have a propensity for inducing
pleural plaques. [38] Methodologies used to analyze pleural and/or parenchymal
tissue for the presence of fibers and association of pleural changes differ
markedly between investigators, however. Human studies of individual exposed
to asbestos fibers are difficult to interpret in regard to toxicity solely related
to fibers <5 µm in length because exposure situations almost uniformly
contain a broad distribution of fiber diameters and length.

Preliminary results of residents of Libby, Montana that were exposed to asbestiform
tremolite indicate a high propensity for pleural changes in comparison to interstitial
changes. [39] There is some indication that exposure to tremolite fibers with
relatively low aspect ratios in comparison to the asbestiform type tremolite
may be capable of causing pleural plaques. [40] Pleural plaques can occur with
minimal exposure to asbestos and can occur within a wide range of tissue burdens
of asbestos fibers which overlap with control populations. [41]

Conclusions

Even though there are no human studies solely of MMVF <5 µm in
length, the available morbidity and mortality studies of MMVF production workers
indicate limited overall toxicity from MMVF exposure.

There are no human studies regarding exposures solely to asbestos fiber
<5 µm in length but there has been some speculation that durable
fibers <10 µm in length and <0.1 to 0.4 µm in diameter may
be associated with pleural plaques in relatively low concentration, in particular
the amphibole tremolite.

For asbestos fibers <5 µm in length, it would appear that very
high doses may have the propensity to cause interstitial fibrosis, particularly
if the fibers are durable within intracellular fluids.

Thresholds for Toxic Action

For asbestos and MMVF less than 5 µm in length, thresholds for toxic
action in humans have not been established but most likely is substantially
higher than the thresholds for long durable fibers with increased aspect ratios
of respirable size.

Asbestos Versus MMVF

Based on animal and human studies, natural occurring asbestos fibers that are
of respiratory size, long and thin with high aspect ratios, and durable within
physiologic fluids represent the highest risk for malignant (lung cancer and
mesothelioma) and non-malignant (interstitial fibers) respiratory disease. These
abnormalities have not been demonstrated in MMVF manufacturing workers.

Dr. McConnell’s Post-Meeting Comments

Background: There have been numerous studies of the effects
of various types of asbestos (ATSDR, in press) and SVFs (ATSDR, in press) in
animals. Both fibrous and nonfibrous particulates have been used. Most studies
have been conducted in rats and hamsters, but others, including nonhuman primates
have been used. Routes of exposure have included inhalation (whole-body and
nose-only), intratracheal instillation, intrapleural implantation/injection,
intraperitoneal injection and ingestion. All of the routes of administration
have their strengths and weaknesses (advantages, disadvantages and limitations)
for use for assessing potential health effects in humans (McConnell, 1995).
However, the inhalation route appears to produce the most relevant data because
it is the only route that duplicates all aspects of human fiber exposure and
disease (inflammation, fibrosis, lung cancer and mesothelioma) resulting from
the exposure (McClellan et al., 1992). Also, the neoplastic changes typically
occur late in the rodents’ life, similar to what occurs in humans exposed to
asbestos. Other routes of exposure are also useful for comparing the toxic potential
of various types of fibers and understanding the mode of action and many of
the mechanisms of fiber toxicity and carcinogenicity. Additionally, the oral
route (ingestion) appears to be the most appropriate route of exposure for studying
the potential hazard of ingested asbestos.

Cancer effects: Rats and hamsters are the most frequently
used species for assessing the potential carcinogenic effects as asbestos (IARC,
1987) and SVFs (IARC, 2002) and have been used with various routes of exposure.
Of the two species, the rat appears to be the most appropriate one because it
exhibits both lung cancer and mesothelioma in response to inhalation of known
human carcinogenic fibers, e.g. asbestos. The hamster can be a useful model
if one is only interested in the inflammatory, fibrogenic and mesotheliogenic
effects of particulates. However, the hamster does not develop lung cancer after
exposure to high levels of either chrysotile (McConnell et al., 1995) or amosite
asbestos (McConnell et al., 1999). Other species have been used but have significant
limitations that preclude their general use for carcinogenic bioassays. For
example, the mouse is not as useful as the rat or hamster because its terminal
airways are smaller and therefore, particulates of a mean mass aerodynamic diameter
(MMAD) of greater than >0.5 um cannot reach the deep lung (alveolar region)
which is the site of primary disease. Non-human primates would be an ideal animal
model but are precluded because of their long life-span (would require at least
20-30 years to demonstrate a noncarcinogenic effect), availability (a cancer
bioassay requires >200 animals/sex), and expense (such a study would cost
>$20 million.

Most chronic rodent inhalation bioassays of asbestos have been conducted in
rats, have not shown significant strain differences and males and females are
equally sensitive to its carcinogenic effects (ATSDR, in press). The only large
series of studies of various types of asbestos showed that if there is a gender
difference, males might be slightly more responsive (Wagner, et al., 1974).
Therefore, either sex is appropriate with males slightly more preferable. Just
as importantly, both sexes are probably not necessary. However, these same studies
have shown that while life-time exposure to asbestos may not be necessary, it
is important to observe the animals for most of their life-span (see below).

The types of cancer induced by asbestos and SVFs in rodents are comparable
to those observed in humans, although the preponderance of a given type and
its biologic behavior appears to be species specific. In inhalation studies
in rats the preponderant form of lung cancer is bronchoalveolar in origin, arising
from type II alveolar cells. They occur late in the animal’s life, usually after
21 months of age. This is why lifetime studies may be necessary to fully exonerate
a fiber from being considered carcinogenic. The tumors are slow growing and
only occasionally are the cause of death. The biological sequence of growth
is typically from bronchoalveolar hyperplasia to bronchoalveolar adenoma to
bronchoalveolar carcinoma, although all aspects of the sequence of progression
may not be found in a given lesion (Boorman and Eustis, 1990). Squamous cell
metaplasia is not unusual and typically is found as part of the morphology of
larger tumors. Squamous cell carcinoma may predominate in a small percentage
of rodent tumors, but has rarely been observed to occur de novo. Squamous cell
types may be more common with intratracheal instillation of the fibers (Pott
et al., 1994). The malignant tumors are locally invasive and can metastasize
but it is an unusual event for them to do so. When this occurs it is usually
within the lung, but distant metastases have been observed. The presence of
mitotic figures is in direct relation to the degree of malignant transformation.
Tumors of the upper respiratory tract and airways have not been observed in
response to inhalation exposure of asbestos or SVFs in rodents (IARC, 1987;
2002).

Mesothelioma has also been found in rodent carcinogenic bioassays of asbestos
and SVFs (IARC, 1987; 2002. In inhalation studies in rats they are usually found
at a lower incidence than lung cancer. Again, there does not appear to be a
gender predisposition and the mesotheliomas in rodents typically occur late
in life (after 21 months of age). They rarely are the cause of death. They grow
by expansion, growing over the pleural surface. They typically do not invade
the lung or other adjacent structures, although this has been observed. They
usually present as multiple lesions on both sides of the lung and involve both
the visceral and parietal pleura. Rarely, distant metastases have been observed.
In inhalation studies, all of the major morphological types (tubulopapillary,
sarcomatous and mixed) have been observed, although the tubulopapillary response
is the predominate form. There is one exception to this and that is found in
the inhalation study of erionite, where the sarcomatous type predominated, was
highly invasive and the tumors were exceptionally lethal causing death in most
of the rats by 15 months (Wagner et al., 1988). In contrast to inhalation, direct
instillation into the pleural (Stanton et al., 1981) or peritoneal cavities
(Pott et al., 1987) results in a preponderance of sarcomatous neoplasms, and
in fact, it may be difficult to find mesothelial cells in many of the tumors,
particularly after peritoneal injection. However, even in these studies, the
mesotheliomas seldom invade local tissues or metastasize to other areas of the
body.

The biological sequence of events in the development of mesothelioma in rodents
also appears to have a series of progressive steps (Boorman et al., 1990). In
inhalation studies, the first event that is observed is fibrosis in the pleura
immediately subjacent to the mesothelial lining. This is multifocal in nature,
possibly occurring more frequently in the interlobular pleura. In the few studies
where the parietal pleura has been investigated (McConnell, et al., 1999), the
initial change was found in the nonmuscular portion of the diaphragm and over
the ribs (as compared to intercostal). The first indication of mesothelial change
is found in these areas of pleural fibrosis. The mesothelial cells become cuboidal
(as compared to a normal squamous morphology) and progress to focal hyperplasia
of one to three cell layers thickness. The next step is the formation of papillary
forms of growth and overgrowth of adjacent pleura. It is at this stage that
mesothelioma is diagnosed. Pseudovacuolated tumor cells may be noted at this
stage. Finally, the tumor evolves into the classical forms noted above. The
course of events is somewhat different for instillation and injection studies.
The initial response in the latter studies is inflammation, followed by a fibrogranulomatous
reaction (assumed to be an attempt to wall off the fibers). A similar sequence
of progression is assumed but results in a higher proportion of sarcomatous
types of mesothelioma.

Pulmonary interstitial fibrosis (see below for description) is invariably found
in studies where either asbestos or SVFs have caused either lung cancer or mesothelioma
(Greim et al., 2001). However, there have been fiber studies where pulmonary
fibrosis was observed without the development of fiber related neoplasms (McConnell,
et al., 1994).

In vitro studies may not be of high value for predicting the carcinogenic potential
of a given type of fiber, although they can give some incite into the difference
between the carcinogenicity of long and short fibers. There are several reasons
for why they may not as useful for predicting the carcinogenic activity of a
given type. First, the fiber used is not subjected to physiological processes
such as clearance and dissolution that are found in the lung. Also, the in vitro test systems use “fresh” fibers so do not typically take into account pathology
attenuating changes in fibers that occur over time in the lung. Finally, the in vitro “dose” may have no relevance to the lung fiber burden. However, not
withstanding this, in vitro methods are highly powerful tools for understanding
fiber/cell interactions and mechanisms of toxicity/carcinogenicity (see Mossman
for details).

Non-cancer effects: Animal models have also demonstrated many
of the same pathological responses that are found in humans exposed to particulates
(IARC, various volumes). The major noncancer endpoints that have been described
in animals in experimental studies are phagocytosis, inflammation and pulmonary
fibrosis. In regard to these endpoints, the rodent lung (and presumably other
species) reacts to asbestos and SVFs as it would to any inhaled nonorganic foreign
body that is not chemically toxic, e.g. beryllium. The lung can only react to
such materials in a limited number of ways. In animals, if the particulate were
deposited in the upper respiratory tract, one would assume that it would be
possible for it to cause local irritation. However, this has not been observed
in inhalation studies, even at high exposure levels. It is assumed that the
resident time for such particles is brief, not allowing for a pathologic response.
The mucous layer in these tissues is relatively thick compared to the size of
the particulate and the methods of removal are quite efficient. The same is
true for the major airways. In experimental animals the airways are intact and
have not been compromised by other toxicants as in humans, e.g. smoking. Therefore,
particulates deposited on these surfaces are again efficiently removed via the
mucociliary escalator and are either swallowed or expectorated. In either case,
the resident time in the body is relatively brief.

For a particulate to cause pathology in experimental animals after inhalation,
it must reach the alveolar region of the lung. Particulate size dictates whether
this happens or not. If the particle reaches terminal bronchiole it causes a
foreign body reaction which is dictated by dose, particle (fiber) size and to
some extent physical chemistry. The lungs’ initial response is an attempt to
remove the offending substance. This is accomplished by resident macrophages.
If the particle is of a size that the macrophage can engulf (phagocytize), it
will be “captured and removed from the lung either by translocation to the airways
or draining lymphatics. As the dose (number of particulates) increases, more
macrophages are recruited. However, if the dose is too large for the number
of available macrophages to remove, an “overload” situation develops which results
in other pathologic events. Such events have been documented in animals both
by histopathology and physiological tests (see Oberdorster for details). If
the fiber is too large to be phagocytized and removed, i.e. longer than the
size of the macrophage [~13 um diameter in rats and hamsters, monkeys ~15 um,
and humans ~21 um diameter (Krombach et al., 1997)], the fiber cannot be removed
unless it is broken into shorter lengths or dissolves (Maxim and McConnell,
2001). Both of the latter two phenomena have been observed with several SVFs
(see below).

If the dose overwhelms the physiological pulmonary defenses or the fiber is
too large to be removed, the initial series of events in animals occur at the
junction of the terminal bronchioles and proximal alveolar duct (this is where
most of the fibers are initially deposited - It should be noted that rodents
do not have a respiratory bronchiole, as do humans). In addition to a stimulating
the local macrophages, an influx of additional macrophages is recruited to the
area. At this point, the local type II alveolar cells (in the proximal alveoli)
undergo metaplasia to a cuboidal appearance and become hyperplastic. The resulting
lesion has been termed “bronchiolization” because the change mimics the appearance
of the terminal airways. Increased amounts of mucous production and sometimes
inspissation of the material often accompany this. Coincident to the bronchiolization,
microgranulomas are observed. These appear to form from a coalition of macrophages
and fibroblasts. At this time the microgranulomas are restricted to the proximal
portion of the alveolar duct, particularly along the alveolar duct ridge. With
time and continued insult the process proceeds peripherally and becomes more
apparent. If the offending fiber persists, collagen is laid down in the adjacent
interstitium (presumably by direct invasion of the fiber into the epithelium
and interstitium). At this time the lesion is referred to as interstitial fibrosis.
In rodent studies, the fibrotic areas are initially focal and widely disseminated.
But, if the insult persists or the dose is high enough, fibrosis becomes more
widespread. Various schemes have been developed to describe these events and
grade them as to their severity for comparative purposes (McConnell et al.,
1984: 2002). There is one notable difference between the qualitative appearance
of the lesions produced by asbestos and SVFs in animals. Neutrophils are often
a prominent part of the inflammatory reaction with asbestos, especially with
amphiboles, while they are rarely found in studies of SVFs, even at doses that
produce fibrosis. The inflammatory reaction can also be documented and quantified
by using the results of pulmonary lavage studies (see Oberdorster).

Stop studies (where exposure is stopped and the animals are observed during
a nonexposed recovery period) have proved useful for determining the reversibility
of the above lesions. Such studies have clearly shown that the initial changes
(macrophage response and bronchiolization) are totally reversible with most
SVFs and to some degree with asbestos. Microgranulomas become less apparent
and early fibrosis is also, to some degree, resolvable, at least with SVFs.
In rodents, studies have demonstrated that fibrosis, even with asbestos, is
not particularly progressive, once the exposure ceases.

While there is no exact correlate for pleural plaques in animals, localized
acellular fibrotic changes reminiscent of this lesion in humans have been observed,
albeit on a much smaller scale. The qualitative changes in the pleura are somewhat
different than in the lung. Macrophages and inflammatory cells are almost totally
absent in the pleural response. Lavage studies have not been conducted with
pleural instillation or peritoneal injection studies so it is not known if the
same events occur with these routes of exposure of exposure. Animal inhalation
studies also suggest that fibers need to be present in the pleura for pathologic
events to occur.

In vitro studies of mesothelial cells have been conducted using both
human and animal cells. These have been primarily designed to study the mechanisms
of carcinogenicity (see Mossman).

Irritant effects: While there is evidence of dermal and ocular
irritation of humans as a response to exposure to asbestos and SVFs, no such
evidence has been observed in animals. Histopathological studies of the nasal
cavity in rodents exposed via inhalation have not shown any evidence of pathology,
although an increased mucous response could be missed with standard histopathology
techniques. Similarly, ingestion studies in rats and hamsters of asbestos did
not reveal any irritation of the alimentary tract (ATSDR, in press).

In vitro studies on the irritant effects of either asbestos or SVFs
in animals have not been reported.

Association between fiber length and fiber-like toxicity: There are numerous animal studies that demonstrate the influence of fiber length
and pathogenicity/carcinogenicity. The early studies by using intrapleural implantation/instillation
(Stanton et al., 1981) and intraperitoneal injection (Pott et al., 1976) in
rats clearly show a direct relationship between fiber size and carcinogenic
activity. The longer the fiber, the more carcinogenic it was in these studies.
These same studies provided the basis for the hypothesis that short fibers,
i.e. shorter than 8 um in length may not represent a significant carcinogenic
risk. However, the same investigations, particularly the intraperitoneal studies
also demonstrated that if the dose was high enough even so-called “innocuous”
particulates, e.g. titanium dioxide, caused the induction of peritoneal mesotheliomas,
albeit at a lower incidence than long fibers. Additionally, the latter studies
also demonstrated that if even long fibers, e.g. wollastonite and some SVFs,
were not carcinogenic if they were not biopersistent in the peritoneal cavity.
There have been a few inhalation studies have been conducted to study the influence
of the fiber length on the pathology of asbestos, and all have been persuasive
for showing that short fibers are not carcinogenic. This has been demonstrated
for chrysotile (Davis and Jones, 1988; Ilgren, 1998; Wagner et al., 1980), amosite
(Davis et al., 1987; 1986) and crocidolite (Davis et al., 1978; Wagner et al.,
1984).

Other circumstantial evidence for considering fiber length as being critical
to the carcinogenic potential of fibers is provided by the observation that
amorphous silica has been shown to be noncarcinogenic in several inhalation
studies in rats, while some types of glass fibers of similar chemistry have
shown to have carcinogenic activity (IARC, 1987). In fact, amorphous silica
has been used as a “negative control” in rodent inhalation studies. A final
piece of evidence for the importance of fiber length for the carcinogenic of
asbestos and SVFs is found in the hilar lymph nodes that drain the lungs of
animals exposed via inhalation to both asbestos and SVFs. These lymph nodes
are literally filled with macrophages containing short fibers and fiber fragments
with no evidence of pathology or neoplastic change in either the lymph nodes
or adjacent tissues.

To summarize studies in animals of short fibers and nonfibrous particulates
have shown that both are potentially carcinogenic if they are introduced into
a confined cavity, e.g. pleural or peritoneal, at sufficiently high doses. But
the same studies clearly show that the carcinogenic potential is definitely
less with fibers of the same type that are longer. Inhalation studies have clearly
shown that short fibers have not caused cancer in animals. The other part of
the equation that needs to be considered is the influence of pulmonary clearance
and biopersistence on the carcinogenic potential of particulates. As noted above,
even long fibers are not carcinogenic in animals unless they are biopersistent
in the animal.

There are only a few in vitro studies that address this subject but
those that have clearly show a relationship between fiber length and genetic
damage. For example, in a study of Chinese hamster ovary cells (CHO) short amosite
failed did not cause chromosomal aberrations while long fiber amosite did (Donaldson
and Golyasnya, 1995). See Mossman and others for other studies.

Thresholds of toxic action: There have been very few inhalation
studies in animals of either asbestos or SVFs to assess a carcinogenic dose
response. It needs to be remembered that to assess a carcinogenic dose response,
one must have a multidose study that shows a carcinogenic response. Most asbestos
and SVF studies were designed to address the carcinogenic potential of the fiber,
not dose response. The only multi-dose inhalation study of asbestos used amosite
in hamsters (McConnell et al., 1995). In that study, there was a definite dose-related
response with regard to both nonneoplastic (macrophage response, pulmonary fibrosis,
etc.) and carcinogenic activity (mesothelioma). Unfortunately, the potential
lung cancer response could not be assessed because hamsters do not develop pulmonary
tumors with particulates. There are a few inhalation studies of SVFs that address
dose response. The only one that was positive for cancer involved refractory
ceramic fibers in rats (Mast et al., 1995). In that study there was a clear
dose response for both cancer and noncancer endpoints and a no-effect level.
There are a few other multidose studies in rats using various types of SVFs,
but since none showed carcinogenic activity, one can only evaluate the dose
response for noncancer endpoints (Hesterberg et al., 1996). Again, there was
evidence in these studies of a dose-related change in the endpoints showing
recognizable change. The “stop-studies” in many of these inhalation studies
(both asbestos and SVFs) provide evidence for a dose response for noncancer
endpoints. However, the number of animals evaluated in the “stop studies” is
too small to address a cancer dose response. The only study in primates that
addresses a potential threshold of action was with chrysotile asbestos (Patek
et al., 1985). In this study, monkeys were exposed to chrysotile asbestos at
an exposure level of 1 mg/m3 (0.8 f/cc >5 um length) for 18 months. Ten months
following the last exposure, lung biopsies were taken and evaluated for fiber
burden and histopathology. There was no evidence of pathology although a few
asbestos bodies were observed in the lung. The monkeys were then held unexposed
for an additional ~11 years at which time they were subjected to necropsy examination
and the lungs for histopathology examination. Again, there was no evidence of
pulmonary pathology and the number of asbestos bodies had decreased (not reported
— personal observation).

In summary, the totality of available data suggests that there is a dose-response
for both neoplastic and nonneoplastic endpoints in animals and there is a no
effect level for both asbestos and SVFs. One attempt at deciding if a given
exposure in animals is potentially carcinogenic involves the use of noncancer
endpoints. In this scheme it was assumed that a dose that caused pulmonary fibrosis
could also represent an exposure that was potentially carcinogenic in animals.
This was because no animal study has ever produced cancer in the absence of
fibrosis. The next assumption was that since no inhalation study had ever shown
fibrosis in the absence of inflammation, one could assume that an exposure that
didn’t result in inflammation would not reasonably be expected to be carcinogenic.
The endpoint chosen for assessing inflammation was the presence of inflammatory
cells over background in bronchoalveolar lavage (BAL) fluid after a 90-day inhalation
exposure. Therefore, if one did not find an increase in inflammatory cells in
BAL fluid, one could chose this exposure as a no-effect threshold.

It is reasonable to expect that in vitro studies could shed light on
the dose response of both asbestos and SVFs. While these types of studies are
primarily designed to capture and elucidate specific mechanisms of toxicity
and carcinogenicity, there may be insights into dose response that could help
in establishing thresholds of effect. One such study showed that short fiber
amosite did not cause inflammation, while long amosite did. The only draw backs
to and in vitro approach is that these techniques do no take lung clearance
phenomena into consideration and fibers that are not biopersistent in the lung
might not be differentiated from biopersistent ones because of the short time
frame of the in vitro studies.

Dr. Mossman’s Post-Meeting Comments

ATSDR Fibers Panel Mechanisms of Short Fiber Toxicity

There appears to be a striking difference in the pathogenicity of respirable
fibers directly related to fiber length, with fibers below approximately 5 microns
in length being less hazardous for the development of cancers or pulmonary fibrosis.
This prompts the questions: What are the observed mechanisms of long fiber toxicity?
Does composition matter? Are short (<5 microns in length) fibers pathogenic?
If so, what are the mechanisms of their toxicity?

One hypothesis is that long fiber effects are related to increased generation
of oxidants; reviewed in Kinnula, 1999; Hansen and Mossman, 1987). It has been
shown that reactive oxygen species (ROS) and reactive nitrogen species (RNS)
are generated by asbestos fibers spontaneously in cell-free systems, cells in
culture, and lung tissue in vivo. A primary step in response to asbestos
fiber challenge to a number of cell types is superoxide anion release from cells
which have attempted to phagocytize long fibers whereas short fibers are encapsulated
in phagolysosomes, often without visible damage to cells. Superoxide, however,
can be further dismutated to hydrogen peroxide which can generate the reactive
hydroxyl radical, catalyzed by iron vs. the Fenton reaction. Alternatively,
superoxide can react with nitric oxide to form peroxynitrite that is associated
with inflammation and lung injury. Asbestos stimulates the release of ROS and
induces oxidants intracellularly in both inflammatory cell types (Hansen and
Mossman, 1987; Goodglick and Kane, 1986; 1990) and target cells (Xu et al.,
2002). Moreover, indirect evidence for oxidant stress by asbestos is indicated
by elevations of antioxidant enzymes in cells in culture and lung tissue after
inhalation of crocidolite asbestos (Janssen et al, 1992, 1994b). In human mesothelial
cells, these increases were not observed with exposures to polystyrene beads,
or riebeckite, a chemically similar nonfibrous analog of crocidolite (Janssen
et al., 1994b). The role of oxidants by crocidolite asbestos in causation of
inflammation and fibrosis has been confirmed in rodent inhalation studies (Mossman
et al., 1990), and supports the central dogma that asbestos fibers activate
transcription factors and early response genes involved in proliferation and
inflammation by generating ROS via “frustrated phagocytosis” (reviewed in Manning
et al., 2002).

Several papers show that “frustrated phagocytosis” and oxidant production occur
selectively in response to long vs. short fibers of asbestos or glass. A study
of luciginen-dependent chemiluminescence (CL) in human monocytes found a strong
correlation between superoxide release and fiber lengths from 6 to 20 microns.
All samples of fibers except wollastonite induced CL release in a dose-dependent
manner. Superoxide release was non-specific for the compositional type of fiber,
and fibers with lengths below 7 microns were only weakly active. In studies
by Blake et al. (1998), CL induction after zymosan stimulation and LDH release,
a measure of lytic cell death, were measured in Manville Code 100 (JM-100) fiber
challenged rat alveolar macrophages. A novel feature of this study was the use
of fibers carefully sized to average lengths of 33, 17, 7, 4, and 3 microns.
The greatest toxicity was seen with the longer fibers which had multiple macrophages
attached along the surface, indicating that incomplete phagocytosis was associated
with toxicity. These studies reinforce the many experiments in the literature
showing that long fibers are more toxic than shorter fibers in a number of cell
types, i.e., Goodglick and Kane, 1990.

Increased fiber length has also been linked to activation of transcription
factors and cytokines. For example, Tumor Necrosis Factor-alpha (TNF) is a cytokine
involved in inflammation and fibrosis. In a study by Ye et al. (1999), glass
fibers with lengths of 6.5 +/- 2.7 microns and 16.7+/-10.6 microns were used
to challenge a mouse macrophage cell line. Glass fibers stimulated TNF production
and caused Nuclear Factor- kB (NF- kB)
activation, a process involving ROS. Long fibers were more potent than short
fibers which were effectively engulfed by macrophages. Short fiber-induced TNF
and TNF gene promoter activation was on the order of one-third to one-half of
long fibers. In another study (Cheng et al., 1999), crocidolite asbestos caused
parallel increases in TNF production in macrophages in a dose-dependent manner,
without cytotoxicity at the optimum stimulating condition. Titanium oxide dust
was without effect. TNF production may also be linked to inflammation by asbestos,
and it has been shown that injection of long vs. short amosite fibers intraperitoneally
results in inflammation and macrophage activation related to the proportion
of long fibers (Donaldson et al., 1989).

Another pathway leading to activation of protooncogenes (fos/jun)
that comprise the Activator Protein-1 transcription factor is the Mitogen Activated
Protein Kinase (MAPK) cascades, consisting of c-jun-N-terminal amino kinases
(JNKs), Extracellular Signal Regulated Kinases (ERKs) and p38 kinases. In studies
by Ye et al. (2001) using macrophages, long glass fibers were more potent than
short fibers in activating MAPK which led to activation of c-Jun and the TNF
promoter. Studies by Zanella et al. (1996) explored the stimulation of ERKs
in mesothelial cells, and found increases with crocidolite and chrysotile asbestos
, but not with the nonfibrous analogs, riebeckite or antigorite. Similarly,
elevations in c-fos and c-jun expression were seen with asbestos
fibers and erionite in mesothelial cells, but were not induced by a variety
of particulates, MMVF-10 or RCF-1 fibers at comparable concentrations (Janssen
et al., 1994a). Long fibers of crocidolite (> 60 microns) were selectively
associated with phosphorylation of the Epidermal Growth Factor receptor in human
mesothelial cells (Pache et al., 1997), an event not occurring with MMVF-10
or particles. In general, pathogenic dusts such as asbestos or silica, produce
a variety of cytokines from cells and activate a number of transcription factors
through ROS or RNS (Mossman and Churg, 1998; Churg et al., 2000).

Another ramification of transcription factor activation is cell proliferation.
Mechanistic studies using target cells in culture or tracheal explants have
shown that long fibers are more toxic and more apt to cause cell proliferation
than short fibers (Brown et al., 1986; Wright et al., 1986; Marsh and Mossman,
1988; Sesko and Mossman, 1989; Woodworth et al., 1983). These events may be
coupled, as compensatory hyperplasia may result from initial epithelial cell
injury. In studies by Woodworth et al. (1983), epithelial proliferation and
squamous metaplasia were observed with various types of fibers including glass
and attapulgite, but not with nonfibrous analogs of asbestos, i.e. riebeckite
and antigorite and other particles.

An intratracheal model in rats using long (>2.5 microns) and short crocidolite
asbestos after intratracheal instillation has yielded some mechanistic information
on the differential effects of long vs. short fibers (Adamson and Bowden 1987a,
b; 1990). These studies suggest that the increased fibrogenic response to long
fibers may be due to selective increases in cell proliferation. In addition,
both long and short asbestos fibers cause alveolar macrophages to secrete fibrogenic
cytokines, but interstitial fibroblasts exposed to short asbestos fibers do
not respond to these cytokines.

Surfactant adsorption may be a mechanism whereby reactive particles or fibers
are rendered inactive or nonpathogenic. To determine the effect of surfactant
adsorption on chrysotile genotoxicity using an assay for micronucleus induction
in Chinese hamster lung cells (V79) (Lu et al., 1994), two lengths of chrysotile
fibers were used with and without pretreatment with DPPC, i.e. NIEHS intermediate
(65%> 10 microns) and short (98% < 10 micron) fibers. The longer fibers
were most active, and DPPC treatment diminished the activity approximately 15%.
The maximum activity of the short fiber sample was 70% of the activity of the
non-treated intermediate, and the DPPC-treated short fibers expressed about
45% of the activity of the untreated. That is, DPPC did not fully suppress the
activity of the fibers, but had a much more pronounced effect on the short fibers.
One possibility is that the partial suppression of genotoxicity reflects suppression
of a component of toxicity by surfactant on the mineral surface. Thus, short
fiber genotoxicity, as reported here, may reflect a combination of mineral surface
functional groups which direct membranolytic activity and can be modulated by
interactions with components of the pulmonary surfactant system as well as phagocytosis-associated
ROS.

In conclusion, studies summarized above show decreased or no effects of short
fibers and nonfibrous analogs of asbestos in a number of bioassays. The effects
of long glass and asbestos fibers may be comparable in some studies. However,
the duration of these short-term assays may be too short to reflect important
solubility changes occurring in lung over time.

Adamson IYR, DH Bowden. (1990) Pulmonary reaction to long and short asbestos
fibers is independent of fibroblast growth factor production by alveolar macrophages.
American Journal of Pathology 137:523-529.

Dr. Oberdörster’s Post-Meeting Comments

When responding to the charge questions in Topic Area 1 (physiological fate
of asbestos and SVF fibers less than 5 micrometers in length), Dr. Oberdörster
gave a brief presentation to the panel. He asked that a copy of the overheads
from this presentation be included in this appendix of the report. A copy of
the overheads Dr. Oberdörster prepared for the meeting follow, including
some overheads that were not shown at the meeting due to time constraints.

Dr. Oberdörster also provided an additional comment not mentioned at the
expert panel meeting. He noted that the panelists overlooked an important concept
of short fiber toxicity which involves an increased retention in the lung of
short fibers in people (e.g., smokers) who have disturbed alveolar macrophage
mediated lung clearance. These people, he noted, can experience a marked increase
in short fiber retention and thereby increase the potential for fiber toxicity
significantly. Long fibers are reportedly not affected to the same degree as
short fibers, as was described in a paper by Churg (“Effects of cigarette smoke
on the clearance of short asbestos fibres from the lung and a comparison with
the clearance of long asbestos fibres,” International Journal of Experimental
Pathology 73(3): 287-297, 1992).

Main Depostion Mechanisms of Inhaled Fibers

Impaction (abrupt directional changes)

Sedimentation (gravitational settlings)

Diffusion (Brownian motion)

Interception (long fibers)

From: Asgharian and Yu, 1988

Deposition Efficiency of Fibers in Different Generations of the Weibel Lung
Model at a Flow Rate of 375 cm3 /sec for dem =0.01µm
and Unit Density.

From: Asgharian and Yu, 1988

Deposition Efficiency of Fibers in Different Generations of the Weibel Lung
Model at a Flow Rate of 375 cm3 /sec for dem = 1 µm
and Unit Density.

From: Asgharian and Yu, 1988

Deposition Efficiency of Fibers in Different Generations of the Weibel Lung
Model at a Flow Rate of 375 cm3 /sec for dem = 10 µm
and Unit Density.

In addition: Clearance is determined by fiber specific physicochemical
processes

Together, these mechanisms define theBiopersistenceof a Fiber

*normal AM-mediated clearance:

T1/2 rat ~70 days

T1/2 human ~400 - 700 days

Pathogenicity and Fiber Length:
The Role of Alveolar Macrophage (AM) Size

Hypothesis:

Phagocytizable fibers

efficient clearance and
prevention of target cell interaction

Average AM Diameters:

Rat:

10.5 — 13 µm

Crapo et al., 1983; Lum et al., 1983, Stone et al., 1992;

Human:

14 — 21 µm

Sebring and Lehnert, 1992; Krombach et al., 1997

For cancer

number of fibers longer than 20 µm

For non-cancer

all fibers (but: also impact for tumors!)

Size Dependent Lymphatic Clearance of
Amosite Asbestos

Study: Intrabronchial instillation of amosite in dogs, followed by

Analysis of mediastinal lymph node and of lymph collected from
Right Lymph Duct

Max. Diameter

Max. Length

Lymph node

0.5 µm

16 µm

Post-nodal lymph

0.5 µm

9 µm

(Oberdörster et al., 1988)

Biopersistence = Biodurability + Physiological Clearance

(Oberdörster, 1996)

From: Timbrell, Inhaled Particles V, 1982

FIG. 8. Bivariate distribution of fibres in Paakkila bagging section.

From: Timbrell, Inhaled Particles V, 1982

FIG. 10. Bivariate presentation of fibre retention

Fiber Dosimetry Following RCF-1 Exposure(Gelzleichter et al., 1996)

FIG 2. Relative size distribution of fibers isolated from (A) aerosol, (B)
Day 5 lung, (C) Day 32 lung, (D) Day 0 pleura, (E) Day 5 pleura, and (F) Day
32 pleura. Isobars were calculated based on histogram analysis of the aerosol
cloud and fiber burden data (average of six rats). Median bin values are shown
on the x and y-axes. Pulmonary and pleural fiber burdens were normalized to
Day 5 data with areas under the curve equal to 100 on Day 5.

From: Timbrell et al., Inhaled Particles VI, 1988

FIG 5. Relationships when the parameter of fibre quantity is number.

From: Timbrell et al., Inhaled Particles VI, 1988

FIG. 3. Relationships when the parameter of fibre quantity is mass. Linked
data points relate to tissue specimens from the same subject.

From: Timbrell et al., Inhaled Particles VI, 1988

FIG. 4. Relationships when the parameter of fibre quantity is surface area.

Dr. Wallace’s Post-Meeting Comments

Health Effects of Asbestos and Synthetic Vitreous Fibers:
The Influence of Fiber Length

I participated in the Agency for Toxic Substances and Disease Registry (ATSDR)
expert panel on “Health Effects of Asbestos and Synthetic Vitreous Fibers: The
Influence of Fiber Length”, held in New York City on October 29-30, 2002. I
limited my comments to one of the topics which ATSDR requested that the panel
consider: “Topic #2: Health Effects of Asbestos and Vitreous Fibers less than
5 micrometers in length.” My research background has involved some studies of
the surface properties and associated toxicities of respirable silica and silicate
particulate dusts, which may have some indirect relevance to one of the questions
asked of the panel under Topic #2, specifically: “Do the mechanisms of action
of other materials (e.g., larger asbestos fibers, silicates, mineral dusts,
amorphous silica) with potentially similar compositions aid in understanding
small-fiber mechanisms of action?”

Dr. Ralph Zumwalde of NIOSH, who has an extensive background in the epidemiology
of fiber-associated diseases, attended the proceedings as an observer and contributed
information and recommendations concerning the availability of data and the
analyses of epidemiology studies of occupational exposures to fibers.

In this review and revision of my comments on the panel, I also comment on
the question: “Is there indirect evidence for less-than-5 micron fiber induced
adverse health effects?” because of its association with the question of mechanism
and because of some reports of inverse correlations of fiber length with fibrosis
seen in asbestos workers’ lungs.

As discussed in the following review, my evaluation of information presented
and commentary made by and to the panel is that there is a need for focused
and short-term research on short fiber hazard; and that there are new opportunities
for the design of that research.

Question: Do the mechanisms of action of other materials (e.g., larger asbestos
fibers, silicates, mineral dusts, amorphous silica) with potentially similar
compositions aid in understanding small-fiber mechanisms of action?

Respirable crystalline silica particles, which are non-fibrous by any definition,
are strongly pathogenic for fibrotic lung disease, and IARC, the US EPA, and
others have recently evaluated quartz and cristobalite, two crystalline silica
polymorphs, to be carcinogenic (1).

Exposure to these crystalline silica dusts can directly damage cells. Research
suggests that consequent to this damage, there can be intrarcellular generation
of reactive oxygen species and a cascade of events similar to the those evoked
by asbestos fiber (2,3). As depicted by Dr. Mossman and others, that sequence
may lead to the synthesis and release of TNF-alpha or other cytokines which
stimulate near-by fibroblasts to proliferate and to up-regulate their synthesis
and secretion of procollagen into the extracellular space of the pulmonary interstitium.
There the procollagen matures into one or several forms of collagen fibers causing
simple or progressive lung fibrosis.

The initial damage by quartz dust and by cristobalite dust to cells in vitro
has been shown to be associated with the presence of silanols, hydroxyl groups
on the crystalline silica surface. Bolasitis et al. (4,5) showed that calcining,
e.g., heating, quartz resulted in the loss of surface silanols and a parallel
loss of direct membranolytic cell damaging activity. As the dust aged in normal
humidity air, the silanols re-formed on the surface over a period of days, and
toxicity was restored parallel to that restoration. Saffiotti et al. (6) observed
similar behavior with cristobalite. In some circumstances, e.g., sand-blasting
occupational exposures, highly reactive free radical species are formed on the
freshly broken crystalline silica surface; these exhibit heightened toxicity
to cells in vitro in the absence of materials which can react to quench that
activity, and may provide a additional mechanism of heightened toxicity (7).

2. Mineral-specific fibrogenicity: Short-term in vitro bioassays for mineral
particles do not work

Some silicate dusts are cytotoxic in vitro but are not strongly pathogenic
in vivo. Clays, layered alumino-silicates, are not associated with strong fibrogenic
activity in human workplace exposures or in animal model exposure studies (8).
In particular, respirable-sized kaolin clay dust, perhaps the structurally simplest
alumino-silicate clay, is comparable to respirable-sized quartz dust for in
vitro cytotoxicity (9) as measured by short term assays of cell damage, e.g.,
membranolysis, cytosolic or lysosomal enzyme release, or dye-exclusion measures
of cell viability. Therefore, direct short-term in vitro cellular assays do
not distinguish the distinct in vivo fibrogenic potentials of quartz versus
kaolin clay dusts. Because of this, the general prevalence of clays in many
mixed dust exposures prevents the use of short-term in vitro cytotoxicity systems
to predict dust hazard.

3. The first events in particle or fiber interaction with the deep lung
surface:

An important but generally ignored component for physiologically-representative
in vitro bioassays:

a. The environmental interface of the deep lung is surfactant-coated

Particles or fibers depositing in the deep lung respiratory bronchioles or
pulmonary alveoli will first contact the aqueous “hypophase” lining on the terminal
airway and airsac surfaces. This thin layer is coated at the air-liquid interface
with surfactant which acts to reduce the surface tension and physically stabilize
the airspaces (10). The hypophase layer is also rich with micellar dispersion
of surfactant. The surfactant is comprised principally of lipids and lipoproteins.
The major constituents are phospholipids: diacyl phosphatidylcholines. Dipalmitoyl
phosphatidyl choline (DPPC) dispersed in physiological saline provides perhaps
the simplest model of lung surfactant, representing the major surfactant constituent
and generally reproducing the surface tension-lowering characteristics of full
lung surfactant.

b. toxic particles adsorb surfactant and are promptly neutralized

Both quartz and kaolin clay dust particles promptly adsorb DPPC surfactant
from dispersion in physiological saline; this immediately coats the particle
surfaces and prophylactically extinguishes their short-term cytotoxicity (12).
The amounts of surfactant in the alveolar hypophase compared to the surface
areas of respirable mineral dusts and their adsorption isotherms for DPPC suggest
that there is adequate surfactant in the lung to coat and neutralize depositing
particles even in most high dust exposures (13).

c. Restoration of particle toxicity and a possible basis for mineral-specific
fibrogenicity

Subsequent to the suppression by pulmonary surfactant of otherwise prompt cytotoxic
activity, the surfactant-coated particles can be phagocytized by macrophages
and subjected to phagolysosomal enzymatic digestion (14). Cell-free experiments
have correlated the digestive removal of DPPC from quartz and kaolin particle
surfaces by phospholipase A2 enzyme with the restoration of membranolytic activity.
In cell-free tests using pH -neutral acting phospholipase A2 and in limited
in vitro/in vivo tests, quartz is stripped of surfactant significantly more
rapidly that kaolin(15). Cellular in vitro studies have found that macrophage-like
cells in vitro digest quartz- and kaolin-adsorbed DPPC at comparable rates over
a period of about 7 to 10 days with initial partial restoration starting at
3 to 5 days (16). It has not been demonstrated that this de-toxification/re-toxification
process is the mechanism distinguishing quartz and alumino-silicate expression
of toxicity in vivo.

4. Site of particulate-induced fibrogenic activity

Churg et al. (17) briefly discuss the principal site of asbestos activity,
noting the alveolar macrophage is commonly regarded as the crucial effector
cell. This is the background assumption also for most experiments on the cytotoxic
and fibrosis-associated activity of crystalline silica dusts. However, Adamson,
referenced by Churg et al. in a different context, has published a suite of
studies which make a case that it is interactions of silica particles with interstitial
cells which control the stimulation of exacerbated collagen synthesis by pulmonary
fibroblasts, and that the macrophage is responsible for only an inflammatory
response evoking neutrophil influx to the alveolus but not tied to explicit
fibrosis(18). While the mechanism of initial cell damage or stimulation may
differ between silica or silicates and fibers, e.g., ROS from a “frustrated”
phagocytosis mechanism for asbestos and surface silanol hydroxyl membranolysis
by quartz or clay, a parallel analysis to Adamson’s silica study findings might
be considered in researching the site of asbestos action for fibrosis.

5. Possible interferences in short-term bioassays

Oberdörster and others (19) have found that the conventional protocol
for extended-term in vitro cellular assays may inadvertently cause a non-physiologic
surface conditioning of mineral particles which significantly affects assay
results. The use of fetal bovine serum can confer a prophylaxis on silica and
perhaps on kaolin (20), probably due to the mineral surface adsorption of lipo-proteins
from the FBS. That may not represent a physiological situation in the intact
lung in vivo and may interfere with attempts to model the condition of particle
surfaces upon deposition in the lung and resultant effects on their expression
of toxicity in vivo. For purposes of in vitro investigation of fiber or particle
toxicity, this interference might be circumvented, e.g., by excluding serum
from the medium during a short-term period for particle or fiber challenge.

6. Environmental conditioning of particle surfaces can affect their in
vivo pathogenic activity

Even animal model in vivo tests can fail to be predictive in the case of a
cytotoxic and fibrogenic mineral in mixed composition dusts, e.g., quartz particles
in workplace dusts: conventional mineralogical and cytotoxicity assays may not
correlate with short-or intermediate-term in vivo fibrogenic response. Alumino-silicate
surface contamination of quartz particle surfaces can delay for months or perhaps
years the expression of fibrogenic activity. Aluminosilicate or other mineral
occlusion of the underlying host particle can alter the expression of toxicity
in vivo during the bio-persistence of the surface contamination. This has been
seen worldwide in anomalies in the fibrogenicity of coal mine dust exposures
(21). This was clearly demonstrated by LeBouffant et al. (22) by in vitro and
in vivo studies of the fibrogenicity of silica in coal mine dusts and in natural
lightly contaminated sands. More recently, new spectroscopic surface analysis
methods have demonstrated natural clay occlusion of quartz dusts from some workplace
where epidemiology studies had detailed anomalies in disease risk correlation
with conventional measures of dust exposure (23).

a. Conventional assays do not clarify the bases of asbestos or silica particle
toxicity

Churg et al. (17) review highlights and caveats to the general models of asbestos
activity. Some fibers can evoke the responses from ROS generation through the
cascade to increased expression of TNF-alpha, but have not been shown to induce
fibrosis. And asbestos produces fibrosis in some systems without increasing
TNF-alpha expression. Chrysotile contains little iron but is fibrogenic, albeit
not a potent as amphibole. Churg et al. suggest a comparison of asbestos and
silica-induced fibrosis data. Their paper compares the generation of ROS, RNS,
and activation of NF-kB and AP-1, and increased production of TNF-alpha and
other factors and find the dusts to be indistinguishable. In the face of this,
asbestosis and silicosis differ in histopathological appearance: asbestosis
is a diffuse fibrosis and silicosis is in localized nodules. Their conclusion
is that the tabulated responses fail to explain comprehensively how asbestosis
or silicosis develop.

b. Surfactant does not fully suppress all asbestos fiber in vitro cytotoxicity

Asbsetos fiber as well as particulate silicate can adsorb the DPPC and components
of pulmonary surfactant (24). We have briefly researched the effect of surfactant
adsorption on chrysotile in vitro genotoxicity, using an assay for micronucleus
induction in cultured Chinese hamster lung cells (V79 cells) (25): in our test
of two chrysotile asbestos fiber samples, pre-treatment with DPPC in physiological
saline surrogate lung surfactant did not fully suppress a short-term toxic activity
to cells in vitro. NIEHS intermediate length chrysotile asbestos fiber (average
101 micrometer length, 65% > 10 micron) and NIEHS short chrysotile asbestos
fiber (average 11.6 micron, 98% < 10 micron) were tested for micronucleus
induction in V79 macrophage-derived cells for 72 hour challenge +/- DPPC surfactant
pre-treatment of the fibers. For the longer fiber sample, DPPC did not significantly
affect the activity, a numerical reduction of about 20% in the activity was
observed but was not statistically significant. However, DPPC treatment reduced
the shorter-length fiber sample activity significantly, to about half that of
the untreated shorter fiber sample. Similar effects were seen for multi-nuclei
induction and for dye-exclusion viability measure for cell toxicity. No activity
was seen for either sample in a sister chromatid exchange assay.

c. A surface modification which did not affect long asbestos fiber toxicity
in vitro

We also attempted to see if a significant surface modification of chrysotile
without a significant modification of fiber size would affect in vitro genotoxic
activity (26). The NIEHS intermediate length chrysotile asbestos fiber used
above was mildly acid leached to remove near-surface magnesium, but to retain
fiber length. The treatment resulted in a 20% reduction in fiber length in each
of three length categories: <3 micron, 3-10 micron, > 10 micron. Spectroscopic
surface analysis and zeta-potential measurements showed significant reduction
in surface-associated magnesium and in its influence on surface chemistry. However
there was no significant change in measured activity for micronucleus induction
between the treated and non-treated fibers.

d. Is there more than one mechanism of fiber cytotoxicity? Do short fibers
also act as particles?

One interpretation of these two experiments is this: at least two mechanisms
are involved in the initial damage or interaction of fibrous particles with
the lung: a component which is at least transiently suppressed by surfactant
conditioning which significantly contributes to shorter fiber activity, and
a component which is not suppressed by surfactant conditioning, and which is
not affected by one significant modification of surface composition and chemistry,
and which is the principal mechanism for longer fibers. That is, a model which
suggests itself is the combination of the frequently discussed “frustrated phagocytosis”
mechanism for longer fibers, e.g., those which are too long to be fully phagocytized
and internalized by the cell target, and a surface property-mediated toxicity
mechanism for internalized particles or short fibers, i.e., fibers which are
internalized and subjected to conventional phagolysosomal processes.

One possible consequence of “frustrated phagocytosis” of longer fibers is that
the partially invaginated fiber stimulates the cell to release superoxide in
a manner related to the respiratory burst upon normal phagocytosis, or that
superoxide is produced by the cell in response to an autolytic effect of enzymes
or other lysosomal or cytosolic agents released into the annular invagination
of the fiber. The superoxide is then in close approximation with reactive iron
species on the fiber surface in or extending beyond the partially invaginated
fiber to create hydroxyl radical for strongly toxic effects at the cell or neighboring
cells. The paper by M Ohyama et al. (27) provided to the panel presents a difficult
argument against frustrated phagocytosis: The study used luciginen-dependent
chemiluminescence (CL) induced in vitro over a short (2 hour) period, and found
a strong correlation of response indicative of superoxide release with fiber
length 6 to 20 um. All samples except wollastonite induced CL response in a
dose-dependent manner. Superoxide release was non-specific for compositional
type of fiber. The four fibers with lengths below 7 um were only weakly active.
Longer fiber activity correlated with length.

Research on the surfactant suppression and subsequent lysosomal enzymatic restoration
of mineral particle cytotoxicity within a cell, suggests that short fibers which
are fully taken into the cell in a phagosome may express, in part, a cytotoxicity
within the cell after removal of adsorbed prophylactic surfactant. That is,
some part of short fiber toxicity may be related to the mineral surface-specific
mechanism of non-fibrous particulate toxicity.

Those do not exhaust the possible mechanisms for long or short fiber damage
to cells. Asbestos fiber penetrating the cell or cell nucleus may exercise modes
of direct genetic or epigenetic damage. In our above study of surfactant effects
on chrysotile genotoxicity in vitro, a limited investigation using immunofluorescent
kinetochore staining indicated that both clastogenic and aneuploidogenic effects
were associated in similar proportion with the observed micronucleus induction.
That is, fibers may directly or indirectly interact with the spindle mechanism
involved in chromosomal separation during cell division. During mitosis, the
nuclear membrane disintegrates, possibly providing intracellular fibers access
to the genetic material or kinetochores and spindle apparatus.

2. Intracellular response to fiber challenge

a. Long fiber challenge

Whatever the mechanisms of direct fiber damage or stimulation of the cell surface,
some components of the consequent intracellular response have been well-defined.
Mossman and others have detailed the cascade of events following fiber challenge
to pulmonary macrophages or perhaps to other cells. A recent review (3) explicates
the central dogma that damage to or stimulation of the cell by fibers is followed
by an increase in intracellular reactive oxygen species which trigger a cascade
of transcription factor activation leading to the up-regulated production and
release of TNF-alpha or other cytokines. This also was recently the subject
of a NIOSH study by Cheng et al. (28) in which crocidolite with a median fiber
length of 11.5 um challenged lavaged rat AM in FBS-containing medium for 1 to
24 h.. Crocidolite caused parallel increases in TNF-alpha production and NF-kB
activation.in a dose-dependent manner. A titanium oxide control dust had no
stimulatory effect on TNF-a secretion. The report by V Kinnula which was provided
to the panel (29) reviews the possible roles of reactive oxygen species (ROS)
and reactive nitrogen species (RNS) generated by asbestos fiber in cell-free
and cellular and tissue systems. A primary step in response to asbestos “long”
fiber challenge of cells is agreed to be superoxide anion release in cells which
have attempted to phagocytize fibers. This superoxide can further be dismutated
to hydrogen peroxide, which can generate hydroxyl radical, catalyzed by iron
via the Fenton reaction. That hydroxyl radical is extremely toxic and reactive,
but therefore short-lived. There is some contention that fibers stimulate the
release of ROS from inflammatory cells and not target cells. However, asbestos
fiber can generate ROS spontaneously in cell-free systems. This fiber-prompted
production and release of TNF-alpha can stimulate nearby pulmonary fibroblasts
to proliferate and increase pro-collagen synthesis, which is released extra-cellularly
to mature into collagen scarring.

Dr. Baron of NIOSH has been developing a fiber size classifier (separator)
which can permit in vitro or perhaps limited in vivo experiments with sets of
fibers of fairly well-defined length (30). A dielectrophoretic classifier can
separate fibers from an airstream producing about 1 mg/day of a size cut. These
classes of JM-100 glass fibers were recently produced for in vitro toxicology
study:

In recent NIOSH studies by Dr. Castranova and colleagues, these samples were
used in a comparison of “long” and “short” fiber cytotoxicity and of induction
of the cytokine cascade in vitro: Blake et al. (31) used 18 hour challenge of
rat alveolar macrophages in vitro and the lactate dehydrogenase (LDH) release
assay, the 17 micrometer sample expressed about 2 X the activity of the shorter
samples (and also 2X the activity of the 33 micrometer longer sample) on a mass
basis. However, all samples were active well above control levels. The 7 micrometer
fiber set had about 8 X more fibers per gram than the 17 micrometer set, or
about 3 X the linear surface area. Thus, the 17 micrometer long fibers were
on the order of 6 or 7 X more cytotoxic than the shorter 7 micrometer fibers
on a linear surface basis. Similar effects were seen with an assay using chemiluminescent
response to zymosan challenge. And multiple macrophages were seen attached along
the length of the long fibers, suggesting “frustrated” or incomplete phagocytosis
was occurring for longer fibers.

J Ye et al.(32) challenged a mouse macrophage cell line with the 7 and with
the 17 um glass fiber cuts, for 3, 6, and 16 h. Glass fibers stimulated TNF-alpha
production, activation of TNF-alpha gene promoter activity, and activation of
DNA binding activity of nuclear factor (NF)-kB. Reactive oxygen species (ROS)
were involved in the activation and production. Dose was set at 5 fibers per
cell; by that metric the longer fibers were more potent than short fibers by
a factor of about 3. However, on a basis of length of fiber exposed to the cell
or surface area, the activities were about equal for the long and short fibers.
As seen in photomicrographs, short fibers but not long fibers were effectively
engulfed by macrophages. In a subsequent study by Ye et al. (33) it was found
that the long fibers were more potent than short fibers at the same dose of
5 fibers/cell in activating MAP kinases which activate transcription factor
c-Jun which acts on the TNF-a gene promoter through the cyclic AMP response
element and the AP-1 binding site. Again, the activities were comparable for
long and short fibers on the basis of exposed fiber length or surface area.

1. Churg et al. (34) found the grade of interstitial fibrosis asbestosis in
the lungs of a group of chrysotile miners and millers to be directly proportional
to tremolite or chrysotile fiber concentrations, but inversely proportional
to mean fiber length and length-related parameters. Churg et al., (35) graded
fibrosis in the lungs of some shipyard and insulation workers, finding fibrosis
grade to be strongly positively correlated with amosite concentration and negatively
correlated with mean fiber size parameters including fiber length; they suggested
“...these observations again raise the possibility that short fibers may be
more important than is commonly believed in the genesis of fibrosis in man.”
In a study of chrysotile miners and millers, Churg, et al., found pleural plaques
were strongly associated with mean tremolite fiber aspect ratio, but no differences
in mean fiber size, including length, were seen for any other disease studied
(mesothelioma, airway fibrosis, asbestosis, or carcinoma) (36). One member brought
to the panel’s attention a recent publication (37) analyzing fibers in lung
tissue from two groups of former chrysotile miners and millers: the study concluded
that “...fiber dimension does not seem to be a factor that accounts for the
difference in incidence of respiratory disease between the two groups”. It has
been generally speculated that shorter fibers in lung tissue may be the residue
of fibers which were longer when deposited, and disease initiation was due to
the originally long fibers, which were subjected to subsequent in vivo dissolution
or degradation into the observed short fibers (17). This appears to be one plausible
explanation of the inverse correlations reported between fibrosis and fiber
length in human lungs. But this does not limit the research opportunity or imperative,
provided by the seemingly anomalous or “counter-intuitive” results, to address
possible short fiber-associated disease mechanisms.

2. A possible “short fiber” exposure cohort. Dr. Zumwalde of NIOSH suggested
to the panel that a past NIOSH study of 2,302 workers at an attapulgite mining
and milling facility (38) may have involved exposures, in part, to short mineral
fibers. A significant deficit of mortality (SMR = 43, 90% CI 23-76) from nonmalignant
respiratory disease (NMRD) was observed for the cohort; but a statistically
significant excess of mortality from lung cancer was observed among whites (SMR
= 193, 90% CI 121-293), but a deficit occurred among nonwhites (SMR = 53, 90%
CI 21-112). This may present an opportunity for review and re-analysis and a
source for collection of materials for study. NIOSH also is re-analyzing archived
materials available from a past study of asbestos workers in South Carolina.

Question: Are short fibers pathogenic? What should
we do?

1. Review of in vitro toxicology

For non-fibrous particles:
- Non-fibrous mineral particles can be cytotoxic, fibrogenic, and carcinogenic.
- That pathogenicity is mineral-specific.
- Surface characteristics may delay expression of that pathogenic activity in
vivo.
- That pathogenicity is not necessarily reflected in short-term in vitro cytotoxicity
assays.
- The first interaction of particles depositing in the deep lung, namely, adsorption
of the lung lining surfactant, strongly affects mineral particle prompt toxicity.
- The bio-persistence of that surfactant prophylaxis may be a critical factor
in the timing and severity of mineral particle expression of toxicity.
- After expression of the primary toxic event in particle challenge to cells,
the intracellular response may be much similar to the cascade induced by asbestos
or fiber challenge: leading to the induction of pathogenic, e.g., fibrogenic
activity by nearby cells.

For fibrous particles:
- Many studies have found an association of pulmonary fibrosis, cancer, and
mesothelioma with occupational exposures to long fibers, e.g., fibers with length
greater than the dimensions of the target cells.
- Long fibers clearly are cytotoxic in vitro.
- Long fiber cytotoxicity and the initiation of pathogenic processes are generally
considered to be resultant from a “frustrated phagocytosis” mechanism.
- Some studies of fiber burden and disease in tissue from asbestos workers have
shown an inverse correlation of disease with fiber length.
- Those disease-correlated shorter fibers appear in some of the cases to be
mineral specific, e.g., associated with contaminant amphibole more than with
seprentine asbestos.
- Limited in vitro study data suggest that shorter fibers may have a component
of cytotoxicity which is surface associated, perhaps independent of a “frustrated
phagocytosis” mechanism involved in long fiber toxicity.
- In short term in vitro assays, well-controlled for fiber length, shorter glass
fibers can cause intracellular events comparable on a fiber or surface basis
to those associated with longer fiber challenge and with asbestos fiber challenge.

2. Interpretability of short-term in vitro assays

The ability to interpret many in vitro experiments on particle of fiber toxicity
is complicated by the lack of modeling of initial conditioning of particles
in the lung and the time course of expression of toxicity in vivo. Surfactant
adsorption in the lung can dramatically alter the short-term in vitro toxicities
of mineral particles, and may determine if toxicity is expressed in times long
or short compared to clearance.

This surfactant effect and associated delay in toxicity expression does not
appear to be a factor for long fiber asbestos expression of in vitro toxicity.
Whether this is a factor for short fibers is unknown. That is, if short fibers
have a component of toxicity independent of a “frustrated phagocytosis” mechanism
but dependent on a surface-property mechanism then such conditioning and time
delays in expression of toxicity could be critical in the design of experiments
for the detection and analysis of short fiber toxicity by in vitro or short-term
in vivo assay.

3. Research opportunities

a. The ability to collect milligram quantities of well-classified (sized) small
fibers presents the opportunity to do carefully size-controlled in vitro studies
and possibly some (more limited) in vivo studies, e.g., by nose-only inhalation
or tracheal instillation.

b. Epidemiology study results suggest types of fibers which should be compared
and contrasted in such experiments, e.g., short tremolite vs. short chrysotile.

c. A review of epidemiological studies of attapulgite or other short fiber
exposures may provide an identification of other short fiber asbestos and non-asbestos
materials for toxicological study for which human disease epidemiology information
is available for comparison.

d. Preceding the initiation of new toxicology studies, a review of past in
vitro studies might identify the controls for surface conditioning of the test
fibers: were effects of lung conditioning modeled, or were non-physiologic effects
of medium adsorbates possible in past studies?

e. So-designed in vitro toxicology studies of classified short fibrous materials
which have known positive or negative correlations with pathology could be attempted
to determine if there is a short fiber toxicity with a reasonable potential
to initiate disease in vivo, and if that potential is
dependent on fiber mineral type or surface property or morphology.

f. A similar review of short-term in vivo studies might help the design of
methods of challenge and time course for tests of materials selected from the
in vitro study results.

g. Results of the in vitro and in vivo studies would suggest if useful application
could be made to dusts of current concern, e.g., Libby vermiculite or World
Trade Center disaster-associated dusts.